The plasmids used in this work, their main characteristics and construction details, are described in Table S1 . The DNA primers used in their construction are listed in Table S2 . Plasmids were constructed and purified using standard molecular biology procedures with proof-reading Phusion DNA polymerase (Thermo Fisher Scientific), restriction enzymes (Thermo Fisher Scientific), T4 DNA Ligase (Thermo Fisher Scientific), DreamTaq DNA polymerase (Thermo Fisher Scientific), DNA clean and concentrator™-5 kit and Zymoclean™ gel DNA recovery kit (Zymo Research), and GeneElute Plasmid Miniprep kit (Sigma Aldrich) or NZYMidiprep kit (NZYtech), according to the instructions of the manufacturers. The backbone plasmids used in this work were: pGADT7 (Clontech) and pGBKT7 (Clontech), used for yeast two-hybrid assays; pEGFP-C1 (Clontech), and pEF6/myc-His C (Thermo Fisher Scientific), used to generate mammalian transfection plasmids; and pSVP247 (da Cunha et al., 2017 (link)), a derivative of p2TK2—SW2 (Agaisse and Derre, 2013 (link)), used to generate a C. trachomatis expression plasmid bearing CT288 with a double hemagglutinin epitope tag (2HA) at its C-terminus (CT288-2HA). The accuracy of the nucleotide sequence of all the inserts in the constructed plasmids was confirmed by DNA sequencing.
Nzymidiprep kit
Manufactured by NZYTech
Sourced in Portugal
The NZYMidiprep kit is a laboratory product designed for the purification of plasmid DNA from bacterial cultures. The kit utilizes a silica-based membrane technology to efficiently isolate and recover plasmid DNA samples.
Automatically generated - may contain errors
Lab products found in correlation
Zymoclean gel dna recovery kit, by Zymo Research (3 mentions)
T4 dna ligase, by Thermo Fisher Scientific (3 mentions)
Dreamtaq dna polymerase, by Thermo Fisher Scientific (3 mentions)
Restriction enzyme, by Thermo Fisher Scientific (3 mentions)
Geneelute plasmid miniprep kit, by Merck Group (3 mentions)
Pegfp c1, by Takara Bio (2 mentions)
Phusion dna polymerase, by Thermo Fisher Scientific (2 mentions)
Dna clean concentrator 5 kit, by Zymo Research (2 mentions)
Pgbkt7, by Takara Bio (1 mentions)
Pgadt7, by Takara Bio (1 mentions)
Dna clean and concentrator 5 kit, by Zymo Research (1 mentions)
Pmal c, by New England Biolabs (1 mentions)
Pgex 4t 2, by GE Healthcare (1 mentions)
Bovine serum albumin (bsa), by Thermo Fisher Scientific (1 mentions)
Dpni, by Thermo Fisher Scientific (1 mentions)
Nzytaq 2, by NZYTech (1 mentions)
Phusion high fidelity dna polymerase, by Thermo Fisher Scientific (1 mentions)
4 protocols using nzymidiprep kit
1
Plasmid Construction and Purification Protocols
2
Plasmid Construction and Purification for Protein Expression
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The plasmids used in this work, their main characteristics and construction details, are described in S1 Table . The DNA primers used in their construction are listed in S2 Table . Plasmids were constructed and purified using standard molecular biology procedures with proof-reading Phusion DNA polymerase (Thermo Fisher Scientific), restriction enzymes (Thermo Fisher Scientific), T4 DNA Ligase (Thermo Fisher Scientific), DreamTaq DNA polymerase (Thermo Fisher Scientific), DNA clean & concentratorTM-5 kit and ZymocleanTM gel DNA recovery kit (Zymo Research), and GeneElute Plasmid Miniprep kit (Sigma Aldrich) or NZYMidiprep kit (NZYtech), according to the instructions of the manufacturers. The backbone plasmids used in this work were pGEX-4T-2 (GE Healthcare) and pMal-c (New England Biolabs), for recombinant protein purification, and pEGFP-C1 (Clontech) for transfection of mammalian cells. Furthermore, pSVP247 (S1 Fig and S1 Table ), a derivative of p2TK2-SW2 [28 (link)], was the backbone to generate C. trachomatis expression plasmids bearing genes whose transcription is halted by the incD terminator (TincD) and encoding proteins with a double hemagglutinin epitope tag (2HA) at their C-terminus. The accuracy of the nucleotide sequence of all the inserts in the constructed plasmids was confirmed by DNA sequencing.
3
Cloning of sgRNA/Cas9 Expression Vectors
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pSpCas9(BB)-2A-Puro (PX459) V2.0, a gift from Feng Zhang (Addgene plasmid # 62988; http://n2t.net/addgene:62988;RRID:Addgene_62988 accessed on 3 June 2023), was used as the backbone for the construction of all sgRNA/Cas9 expression vectors used in this study [38 (link)]. The PX459-containing Escherichia coli Stbl3 strain (supplied as a stab culture) was handled according to the provider’s recommendations (Addgene, Watertown, MA, USA), and plasmid DNA was recovered using the NZYMidiprep Kit (NZYTech, Lisbon, Portugal) following the manufacturer’s instructions. Before sgRNA cloning, plasmid DNA (5 μg) was digested with 25 U of BbsI in the presence of 1 × buffer G (with bovine serum albumin (BSA)) for 6 h at 37 °C (both from Thermo Fisher Scientific, Waltham, MA, USA), separated by electrophoresis on a 0.8% (w/v) agarose gel, and further isolated with Cut and Spin Gel Extraction Columns (GRiSP, Porto, Portugal) according to the manufacturer’s instructions.
4
Plasmid Construction and Cloning Protocols
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Plasmids and oligonucleotides used in this work are listed in S4 and S5 Tables, respectively, as well as their relevant characteristics. Plasmids were generated using restriction enzymes or by restriction-free cloning [54 (link)]. For cloning using restriction enzymes, plasmids were constructed and purified using standard molecular biology procedures, using Phusion high-fidelity DNA polymerase (Thermo Fisher Scientific), restriction enzymes (Thermo Fisher Scientific), T4 DNA Ligase (Thermo Fisher Scientific), DreamTaq DNA polymerase (Thermo Fisher Scientific), NZYTaqII (NZYTech), DNA clean & concentrator™-5 kit, Zymoclean™ gel DNA recovery kit (Zymo Research), and GeneElute Plasmid Miniprep kit (Sigma-Aldrich) or NZYMidiprep kit (NZYTech) according to manufacturer’s instructions. For restriction-free cloning, plasmids were generated using a PCR-based method [54 (link)] with Phusion high-fidelity DNA polymerase (Thermo Fisher Scientific), and DpnI (Thermo Fisher Scientific) was used to degrade parental plasmids. The accuracy of the nucleotide sequence of all the inserts in the constructed plasmids was confirmed by DNA sequencing.
For the generation of plasmid-encoded Inc-2HA or Inc-GSK fusion proteins, inserts were composed of DNA sequences comprising the inc gene plus 300 base pairs upstream from the transcription start codon to include the promoter region.
For the generation of plasmid-encoded Inc-2HA or Inc-GSK fusion proteins, inserts were composed of DNA sequences comprising the inc gene plus 300 base pairs upstream from the transcription start codon to include the promoter region.
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